Isolation and use fibronectin receptor

ABSTRACT

Method for the isolation and characterization of a 140,000 dalton cell surface glycoprotein with the properties expected of a fibronectin receptor is described.

This application is a divisional of application Ser. No. 08/468,480,filed Jun. 5, 1995, now U.S. Pat. No. 5,766,857, which is a divisionalof application Ser. No. 08/056,815, filed Apr. 29, 1993, now U.S. Pat.No. 5,540,933 which is a continuation of application Ser. No.07/857,097, filed Mar. 20, 1992, abandoned, which is a continuation ofapplication Ser. No. 07/302,047, filed Jan. 25, 1989, abandoned, whichis a continuation of application Ser. No. 06/740,240, filed May 31,1985, abandoned.

FIELD OF THE INVENTION

This invention relates generally to the field of biochemistry and moreparticularly to a cell surface glycoprotein having the apparentmolecular weight of 140,000 daltons with the properties expected of afibronectin receptor, and the use of the receptor to prepare liposomeswith predetermined adhesion properties.

BACKGROUND OF THE INVENTION

Cell-substrate adhesion is generally considered to be a multistepprocess involving recognition of extracellular matrix components by cellsurface receptors, followed by cytoskeletal rearrangements that lead tocell spreading (Grinnell, 1978; Hynes, 1981). Several extracellularmatrix glycoproteins, such as fibronectin (Ruoslahti, et al., 1981b),laminin (Timpl, et al., 1979), vitronectin (Hayman, et al., 1983), andcollagens have been shown to promote attachment of various cell types totissue culture substrates (Kleinman, et al., 1981). The cell membranereceptors that recognize these matrix proteins, however, remainessentially unknown, although putative receptors for laminin (Lesot, etal., 1983; Malinoff and Wicha, 1983) and collagens (Chiang and Kang,1982; Mollenhauer and von der Mark, 1983) are currently beinginvestigated.

A number of candidates for the role of a fibronectin receptor have beenproposed. By photoaffinity labeling, it was shown that a 49 kdglycoprotein comes into close contact with substrate-bound fibronectin(Aplin, et al., 1981). Further support for the notion that the receptoris a protein comes from studies showing that treatment of cells withcertain proteases abolishes the ability of cells to attach tofibronectin (Tarone, et al., 1982). Treatment with trypsin, however, atleast in the presence of Ca⁺⁺, leaves the receptor activity intact(Oppenheimer-Marks and Grinnell, 1984). Based upon the calcium-dependentstability to trypsin, Oppenheimer-Marks and Grinnell (1984) haveproposed a 48 kd wheat germ agglutinin-binding glycoprotein as apotential fibronectin receptor.

It has also been suggested that heparan sulfate proteoglycans might beinvolved in cell attachment to fibronectin (Culp, et al., 1979; Laterra,et al., 1983). Indeed, photocross-linking experiments performed byPerkins, et al. (1979) showed that proteoglycans are associated withfibronectin at the cell surface.

A different type of cell surface component has been implicated infibronectin-cell interactions by studies showing an inhibitory effect ofdi- and trisialogangliosides on the attachment of cells to fibronectin(Kleinman, et al., 1979). The inhibitory activity was found to reside inthe carbohydrate moiety of the glycolipid. Antibodies that interferewith cell attachment have been described by a number of investigators,and the corresponding antigens have been found to be proteins withmolecular weights ranging from 60 to 160 kd (Hsieh and Sueoka, 1980;Knudsen, et al., 1981; Neff, et al., 1982; Greve and Gottlieb, 1982;Oesch and Birchmeier, 1982), or specific gangliosides (Dippold, et al.,1984).

A large number of binding affinities are known to be present in thefibronectin molecule, such as for collagen (Engvall and Ruoslahti,1977), fibrinogen and fibrin (Ruoslahti and Vaheri, 1975), proteoglycans(Stathakis and Mosesson, 1977), cell surfaces (Klebe, 1974; Pearlstein,1976), and actin (Keski-Oja, et al., 1980), and there have been somestudies of the interaction of cell surfaces with the cell attachmentsite. (Pierschbacher, et al., 1981). A large fibronectin fragment, thatpromotes cell attachment but lacks the other binding activities is alsoknown, (Pierschbacher, et al., 1982, 1983; Pierschbacher and Ruoslahti,1984a).

It has now been discovered that a 140 kd protein from detergent extractsof cells, when incorporated into liposomes, promotes their bindingspecifically to fibronectin-coated substrates via the Arg-Gly-Aspsequence in the fibronectin molecule.

REFERENCES

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SUMMARY OF THE INVENTION

The present invention relates to the discovery, identification,separation and isolation, and the use of a cell surface glycoproteincharacterized in that it has a molecular weight of about 140,000daltons, interacts with the cell attachment site in fibronectin and issubstantially separated from interfering and diluting cell surfaceglycoproteins.

As a composition of matter, the invention may be described as consistingessentially of cell surface glycoprotein characterized in that it has amolecular weight of about 140,000 daltons, interacts with the cellattachment site in fibronectin, is eluted from such cell attachment siteby a peptide consisting essentially of the amino acid sequence Arginine,Glycine, Aspartic Acid (ARG-GLY-ASP), and is substantially separatedfrom interfering cell surface glycoproteins.

The composition may also be described as consisting essentially of cellsurface glycoprotein characterized in that it has a molecular weight ofabout 140,000 daltons, interacts with the cell attachment site infibronectin, can be labelled by a procedure which is specific for cellsurface proteins, and is substantially separated from interfering cellsurface glycoproteins.

The invention, in one facet, may be described as a cell surfaceglycoprotein characterized in that it has a molecular weight of about140,000 daltons, interacts with the cell attachment site in fibronectin,incorporates into lipid vesicles, and is substantially separated frominterfering cell surface glycoproteins.

The invention also contemplates a method utilizing a composition fortargeting liposomes to fibronectin-containing tissues consistingessentially of a cell surface glycoprotein having a molecular weight ofabout 140,000 daltons which interacts with the cell attachment site infibronectin and is eluted from such cell attachment site by a peptideconsisting essentially of the sequence ARG-GLY-ASP.

The composition for targeting liposomes to fibronectin containingtissues may, in a preferred form, consist essentially of cell surfaceglycoprotein having a molecular weight of about 140,000 daltons whichinteracts with the cell attachment site in fibronectin, is eluted fromsuch cell attachment site by a peptide consisting essentially of thesequence ARG-GLY-ASP.

The invention, in another feature, is a method of targeting liposomes tofibronectin containing tissues comprising exposing such tissues toliposomes containing cell surface glycoprotein characterized in that ithas a molecular weight of about 140,000 daltons, interacts with the cellattachment site in fibronectin and is substantially separated frominterfering and diluting cell surface glycoproteins.

The preferred method of targeting liposomes to fibronectin containingtissues comprises exposing such tissues to liposomes containing a cellsurface glycoprotein characterized in that it has a molecular weight ofabout 140,000 daltons, interacts with the cell attachment site infibronectin, is eluted from such cell attachment site by a peptideconsisting essentially of the sequence ARG-GLY-ASP, and is substantiallyseparated from interfering cell surface glycoproteins.

The method of targeting liposomes to fibronectin containing tissues maycomprise exposing such tissues to liposomes containing a cell surfaceglycoprotein having a molecular weight of about 140,000 daltons whichinteracts with the cell attachment site in fibronectin, is eluted fromsuch cell attachment site by a peptide consisting essentially of thesequence ARG-GLY-ASP, labelled by a procedure which is specific for cellsurface proteins.

As a new composition of matter suitable for assay and detectionpurposes, the invention is at least one liposome containing a cellsurface glycoprotein characterized in that it has a molecular weight ofabout 140,000 daltons, interacts with the cell attachment site infibronectin, is eluted from such cell attachment site by a peptideconsisting essentially of the sequence ARG-GLY-ASP, and is substantiallyseparated from interfering cell surface glycoproteins.

The liposome composition preferably contains labelled cell surfaceglycoprotein having a molecular weight of about 140,000 daltons whichinteracts with the cell attachment site in fibronectin and is elutedfrom such cell attachment site by a peptide consisting essentially ofthe sequence ARG-GLY-ASP.

As assay reagent, the invention may be described as consistingessentially of a carrier and cell surface glycoprotein characterized inthat it has a molecular weight of about 140,000 daltons, interacts withthe cell attachment site in fibronectin, incorporates into lipidvesicles, and is substantially separated from interfering cell surfaceglycoproteins.

The assay reagent preferably consists essentially of a carrier and alabelled cell surface glycoprotein having a molecular weight of about140,000 daltons which interacts with the cell attachment site infibronectin, is eluted from such cell attachment site by a peptideconsisting essentially of the sequence ARG-GLY-ASP.

The assay reagent may also consist essentially of a carrier and at leastone liposome containing cell surface glycoprotein characterized in thatit has a molecular weight of about 140,000 daltons, interacts with thecell attachment site in fibronectin, incorporates into lipidvesicles,and is substantially separated from interfering cell surfaceglycoproteins. The assay reagent preferably includes cell surfaceglycoprotein having a molecular weight of about 140,000 daltons whichinteracts with the cell attachment site in fibronectin and is elutedfrom such cell attachment site by a peptide consisting essentially ofthe sequence ARG-GLY-ASP. The cell-cell surface glycoprotein can belabelled by a procedure which is specific for cell surface proteins.

The compositions and methods of this invention are not limited to thespecific examples given herein; rather, these compositions and methodsare fundamental tools and will find broad applicability in scientificresearch, clinical assays and therapy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a representation of sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) analysis of fractions eluted from thefibronectin cell-binding fragment affinity matrix. MG-63 humanosteosarcoma cells (10⁸ cells) were surface-labeled and extracted asdescribed in Example I. The extract (2 ml) was chromatographed on acolumn of Sepharose 4B (bed volume 2 ml) containing a covalently bound,cell-attachment-promoting fragment of fibronectin. Fractions of 1 mlwere collected, and aliquots (50 μl) of each fraction were analyzed bySDS-PAGE (7.5% acrylamide) under reducing conditions, usingautoradiography (A) or silver staining (B) for visualization of proteinbands. Where indicated by arrows, 1 mg/ml of the synthetic peptideglycyl-L-arginyl-glycyl-L-glutamyl-L-seryl-L-proline (GRGESP) orglycyl-L-arginyl-glycyl-L-aspartyl-L-seryl-L-proline (GRGDSP) was addedto the elution buffer. Lane 1, flow-through; lanes 2-12, columnfractions obtained by washing the starting buffer alone or supplementedwith peptides; lane 13, material eluted with 8 M urea. Arrowheads denotethe position of the 140 kd protein. Molecular weight markers in this andsubsequent figures were: myosin, 200 kd; β-galactosidase, 116 kd;phosphorylase B, 94 kd; bovine serum albumin, 67 kd; ovalbumin, 43 kd.

FIG. 2 is a representation of the analysis of the 140 kd protein bySDS-PAGE under nonreducing conditions. A preparation of radiolabeled 140kd protein, as shown in FIG. 1, lane 10,and molecular weight markerswere subjected to SDS-PAGE under nonreducing and reducing conditions.The nonreduced and reduced samples were run on separate gels. Lane 1,molecular weight markers visualized using silver staining; lane 2, 140kd protein visualized using autoradiography.

FIG. 3 is a graph depicting incorporation of the 140 kd protein intoliposomes. A 140 kd protein fraction (0.5 ml) obtained by affinitychromatography as shown in FIG. 1 was supplemented with 200 μg ofphosphatidylcholine and 2.5×10⁶ cpm of ³H-phosphati-dylcholine anddialyzed against phosphate-buffered saline (PBS) containing 1 mMphenylmethyl sulfonyl fluoride (PMSP) for 24 hr at 4° C. The resultingliposomes were fractionated by sucrose gradient centrifugation underconditions described herein. Two hundred microliter fractions of thegradient and the pellet (P) were analyzed for ³H-labeled lipid by liquidscintillation counting and for the ¹²⁵I-labeled 140 kd protein by gammacounting. Note that interference by ¹²⁵I in liquid scintillationcounting was negligible, since the ³H radioactivity was present in alarge excess relative to ¹²⁵I.

FIG. 4 is a representation of SDS-PAGE of fractions obtained aftersucrose gradient fractionation of 140 kd protein-containing liposomes.Top and bottom fractions and the pellet of the sucrose gradientfractionation shown in FIG. 3 were analyzed by SDS-PAGE undernonreducing conditions, using silver staining for the visualization ofthe bands. Lane 1, 140 kd protein-containing fraction beforeincorporation into liposomes; lane 2,top fraction (liposomes); lane 3,bottom fraction (nonincorporated proteins); lane 4, pellet. Equalvolumes (100 μl) of sample were applied to each lane. Molecular weightstandards were run in the lanes marked S. Arrowheads indicate theposition of the bands corresponding to the nonreduced form of the 140 kdprotein.

FIG. 5 is a graph depicting binding of the liposomes containing the 140kd protein to fibronectin-coated substrate. ³H-labeled liposomes (5×10⁴cpm/μg phosphatidylcholine) were prepared as described in the legend toFIG. 3. Fifty micrograms of phosphatidyl-choline (PC) and 140 kd proteinfrom 2×10⁸ cells (estimated to be approximately 10 μg based on stainingin SDS-PAGE) were used. One hundred microliters of the liposomesuspension (containing 3 μg phosphatidylcholine) were added tomicrotiter wells coated with various concentrations of fibronectin (FN)or laminin (LM), and the binding assay was carried out as described inExample I.

FIG. 6 is a graph depicting specificity of binding of the 140 kdprotein-liposomes to fibronectin. The liposome-attachment assay wasperformed as in FIG. 5. The protein concentration used in the coatingwas 20 μg/ml. Where indicated, synthetic peptides (1 mg/ml) were addedto the wells with the liposome suspension. The active cell-attachmentpeptide (GRGDSP) inhibits liposome binding by approximately 50%, whileinactive variants have no effect. The mean and range are given for eachcondition.

FIG. 7 is a graph depicting lectin affinity chromatography of 140 kdprotein. ¹²⁵I-labeled 140 kd protein obtained by fibronectin fragmentchromatography was chromatographed on columns (bed volume, 1 ml)containing Wheat Germ-Agglutinin (WGA)-Sepharose or Concanavalin (Con)A-Sepharose. The columns were eluted with PBS containing 0.5 Nonidet-P40and 1 mM PMSF, and supplemented with the appropriate sugar whereindicated by arrows. Arrow 1 denotes the addition of α-methylmannoside(αMM) to the WGA-column and N-acetyl glucosamine (NAGA) to theConA-column, arrow 2 shows the addition of NAGA to the WGA and . KMM tothe ConA column.

FIG. 8 is a representation of SDS-PAGE of the 140 kd protein eluted fromWGA-Sepharose. The fractions obtained as shown in FIG. 7 were analyzedunder nonreducing conditions using autoradiography (A) or silverstaining (B) for the visualization of the protein bands. Lane 1,samplebefore application to the column; lane 2, material eluting in theflow-through; and lane 3, material eluted specifically byN-acetyl-glucosamine.

DETAILED DESCRIPTION OF THE INVENTION

Extracts of surface-labeled cells were fractionated on an immobilizedfibronectin fragment that is capable of promoting cell attachment. Humanosteosarcoma MG-63 cells that had first been surface-iodinated withlactoperoxidase were dissolved in octylglucoside. MG-63 cells attach toand spread on fibronectin, and deposit fibronectin-containingextracellular matrix fibers in a manner similar to that of normalfibroblast cell lines.

An affinity matrix was prepared by coupling to Cyanogen-Bromide(CNBr)-activated SEPHAROSE a 120 kd chymotryptic fragment of fibronectinthat binds to neither gelatin nor heparin but retainscell-attachment-promoting activity (Ruoslahti, et al., 1981a;Pierschbacher, et al., 1981). Specific elution was effected by treatingthe column with the synthetic peptide:

glycyl-L-arginyl-glycyl-L-aspartyl-L-seryl-L-proline

(GRGDSP), which contains the cell-attachment recognition site offibronectin (Pierschbacher and Ruoslahti, 1984a, 1984b). The elutedfractions were analyzed by SDS-PAGE followed by silver staining andautoradiography for detection of total protein and radioactively labeledsurface proteins, respectively.

As shown in FIG. 1A, a radioactive protein with an apparent molecularweight of 140 kd specifically eluted from the affinity matrix with theGRGDSP peptide (lanes 9-12). The affinity of the 140 kd protein for thematrix was essentially unaffected by a related peptide (lanes 4-7) inwhich the aspartic acid residue is replaced by glutamic acid (GRGESP).Since this peptide does not promote cell attachment (Pierschbacher andRuoslahti, 1984b), but does have structural and charge propertitesclosely similar to those of the active peptide, the resistance of the140 kd protein toward elution with this peptide establishes thespecificity of the elution with the active peptide. No specific proteinbands could be obtained by the same elution procedure whenalbumin-SEPHAROSE was used instead of the fibronectinfragment-SEPHAROSE.

Silver-stained SDS-PAGE (FIG. 1B) showed that the 140 kd protein was amajor component among the proteins eluted from the fibronectin fragmentcolumn. It appeared as a darkly stained, diffuse band. The silverstaining also revealed the presence of a number of other protein bandsin the eluted material. The elution of these additional bands, however,was not dependent upon the GRGDSP peptide, and they were not labeled bylactoperoxidase-catalyzed iodination of intact cells. Thus, it appearsthat these bands represent intracellular, possibly cytoskeletal,proteins that bind nonspecifically to the column and slowly leach offduring the elution.

Elution of the affinity column with urea subsequent to the elution withthe GRGDSP peptide resulted in the release of a large number of proteins(FIG. 1, lanes 13). This underlines the high specificity provided by theelution with the synthetic cell-attachment peptide. The urea eluate didnot contain detectable amounts of the 140 kd protein, suggesting that ithad been quantitatively removed from the column by elution with theGRGDSP peptide.

When the ¹²⁵I-labeled, affinity-purified 140 kd protein was subjected toSDS-PAGE under nonreducing conditions, a major band appeared at 120 kd(FIG. 2). In addition, a double band was seen at the position that isoccupied by the single band under reducing conditions. No radioactivitywas found in larger aggregates, indicating that the 140 kd protein isnot cross-linked by disulfide bonds into oligomers. The increasedmobility of the nonreduced protein in SDS-PAGE (120 kd vs. 140 kd)suggests that the molecule has a compact conformation stabilized bydisulfide bonds. The conformation of the 140 kd protein is apparentlyinfluenced by intrachain disulfide bonds.

A preparation containing ¹²⁵I-labeled 140 kd protein dissolved inoctylglucoside (as shown in FIG. 1, lane 10) was mixed withphosphatidylcholine (containing a tracer of ³H-phosphatidylcholine), andthe mixture was freed of detergent by dialysis againstphosphate-buffered saline (PBS). After centrifugation through a sucrosegradient with the sample loaded at the bottom of the tube, thedistributions of the resulting liposomes and of the ¹²⁵I-labeled proteinwere determined. As shown in FIG. 3, approximately 90% of the¹²⁵I-labeled protein codistributed with the tritium-labeled lipidvesicles. SDS-PAGE analysis of the top and bottom fractions and thepellet obtained by the centrifugation (FIG. 4) showed that most of thecontaminating proteins present in the sample remained at the bottom ofthe tube or were pelleted, while the 140 kd protein migrated to the topof the gradient, appearing highly enriched in the liposome fraction.When the sucrose gradient centrifugation was carried out with a sampleprepared without added phospholipid, most of the 140 kd proteinradioactivity was recovered in the pellet fraction and none of itfloated to the top of the gradient. Even when the detergent was notremoved by dialysis, all of the reactivity remained in the lowerfractions of the gradient, but did not precipitate. This indicates thatfloating of the 140 kd protein occurs only in the presence ofphospholipid vesicles and is not due to the presence of residualdetergent bound to the protein. These results indicate that the 140 kdprotein can become inserted into a lipid bilayer.

As depicted in FIG. 5, liposomes containing the 140 kd protein bound tofibronectin-coated microtiter wells in a dose-dependent manner, similarto the attachment of cells to fibronectin. In contrast, no significantbinding to another adhesive glycoprotein, laminin (Engvall, et al.,1983), was observed. In further control experiments, it was found thatneither liposomes without incorporated protein nor liposomes preparedfrom a crude octylglucoside extract of cells adhered tofibronectin-coated wells. In addition, the binding was specificallyinhibited by the cell-attachment peptide, GRGDSP (FIG. 6). This peptide,at a concentration of 1 mg/ml, inhibited the binding of liposomes byapproximately 50%, while inactive variants, GRGESP and GRADSP (alaninereplacing glycine in the latter peptide), were without any effect at thesame concentration. Increasing the concentration of the peptides to 4mg/ml resulted in a higher degree of inhibition, but at thisconcentration some apparent nonspecific inhibition was observed.

Chromatography on WGA-SEPHAROSE or ConA-SEPHAROSE of a fractioncontaining affinity-purified, radiolabeled 140 kd protein showed thatmost of the radioactivity binds to WGA and elutes specifically withN-acetylglucosamine, whereas it does not bind to Con A (FIG. 7).Analysis of the fractions by SDS-PAGE under nonreducing conditionsconfirmed the presence of the radiolabeled 140 kd protein in thefraction eluted with N-acetylglucosamine (FIG. 8A). Silver staining ofSDS-PAGE showed that the WGA-bound material was further enriched for the120 kd and 140 kd components characteristic of the nonreduced 140 kdprotein (FIG. 8B, lane 3) relative to the preparation obtained fromfibronectin fragment-SEPHAROSE (FIG. 8B, lane 1).

EXAMPLE I

Synthetic peptides were prepared by Peninsula Laboratories (San Carlos,Calif.) according to our specifications (Pierschbacher, et al., 1983;Pierschbacher and Ruoslahti, 1984a, 1984b). Fibronectin was preparedfrom human plasma according to Engvall and Ruoslahti (1977). Laminin wasprepared from rat yolk sac tumor according to Engvall, et al. (1983).Egg yolk phosphatidylcholine was purchased from Sigma (St. Louis,Mo.),octylglucoside from Behring Diagnostics (La Jolla, Calif.). ¹²⁵I-sodiumiodide was from Amersham (Arlington Heights, Ill.) and³H-phosphatidylcholine from New England Nuclear (Boston, Mass.).Chemicals used for SDS-PAGE were from BioRad (Richmond, Calif.).

MG-63 human osteosarcoma cells (Billiau, et al., 1977) were grown on 175cm² tissue culture dishes in Dulbecco's Minimum Essential Medium (DMEM)supplemented with 5% fetal calf serum, glutamine, andpenicillin/streptomycin. For subculturing or harvesting, confluentlayers of cells were incubated in 1 mM Ethylenediamine Tetraacetic Acid(EDTA) for 15 minutes.

Cells grown to confluence in 175 cm² dishes were detached with 1 mM EDTAfor 15 minutes, collected by centrifugation and resuspended inphosphate-buffered saline (PBS, 150 mM NaCl, 10 mM sodium phosphate, 1mM CaCl₂, 1 MM MgCl₂, pH 7.3) containing 0.2 mM phenylmethylsulfonylfluoride (PMSF; added from a 100× stock solution in ethanol).The suspended cells were radioiodinated according to Lebien, et al.(1982) using 2 mCi of ¹²⁵I-sodium iodide and 0.2 mg/ml oflactoperoxidase per 10⁸ cells. All subsequent operations were performedat 4° C. Cells were lysed by adding 1 ml of PBS containing 200 mMoctylglucoside and 3 mM PMSF to 10⁸ packed cells and incubating for 10min. Insoluble material was removed by centrifugation at 12,500×g for 15minutes.

The 120 kd chymotryptic cell binding fragment of fibronectin wasprepared and coupled to cyanogen-bromide-activated SEPHAROSE (Sigma) asdescribed (Ruoslahti, et al., 1981a; Pierschbacher, et al., 1981). Thismatrix contained 3 mg/ml of the chymotryptic fragment of fibronectin.

The octylglucoside extract of cells was then applied to 2 ml of thisaffinity matrix, which had been equilibrated in column buffer (PBScontaining 50 mM octylglucoside and 1 mM PMSF). Elution with thesynthetic cell-attachment peptide was carried out by slowly washing thecolumn with 1 volumn column buffer supplemented with 1 mg/ml of GRGDSPover a period of 1 hour.

Samples of SDS-PAGE were boiled for 3 min in the presence of 3% SDS,with or without 5% 2-mercaptoethanol, and electrophoresed on 7.5%acrylamide gels according to Laemmli (1970). Molecular weight markerswere myosin (200 kd), β-galactosidase (116 kd), phosphorylase B (94 kd),bovine serum albumin (67 kd), and ovalbumin (43 kd). Gels weresilver-stained using a commercially available reagent kit (Bio-Rad),following the manufacturer's instructions. Autoradiography was performedby placing Kodak XAR X-ray film between the dried gel and a CRONERLIGHTNING PLUS intensifying screen (DuPont, Newtown, CT) at −70° C. for1-3 days.

Liposomes were prepared essentially as described by Mimms, et al.(1981). Egg yolk phosphatidyl-choline was dried onto a glass tube undera stream of N₂ and dissolved in PBS containing 50 mM octylglucoside orprotein fractions in this buffer. Detergent was removed by dialysisagainst PBS for 24 hours at 4° C., resulting in the formation ofliposomes with an average diameter of approximately 200 nm, as judged byelectron microscopy. To purify the liposomes, the suspension was made45% in sucrose, overlaid with 2 ml of 30% sucrose and 1 ml of 10%sucrose, and centrifuged at 4° C. for 18 hours at 45,000 rpm in aBeckman SW60 rotor. The liposomes were recovered as a white band at thetop of the 10% sucrose layer.

Wells of a polystyrene microtiter plate (Linbro/Titertek, Inglewood,Calif.) were coated with protein solutions in PBS by incubatingovernight at room temperature. Unoccupied binding sites on thepolystyrene surface were then saturated by incubation with 2 mg/mlbovine serum albumin (BSA) in PBS for 2 hours at 37° C. ³H-labeledliposomes suspended in PBS containing 2 mg/ml BSA were added to thewells and incubated for 5 hours at 4°C. The supernatants were thenremoved and wells were washed twice with PBS. Bound liposomes weredissolved in 1% SDS (100 μl/well) and quantitiated by scintillationcounting.

INDUSTRIAL APPLICATION

This invention finds direct and immediate application in the assay offibronectin receptor in cells and tissues. The isolation methoddescribed above can be used to assay cultured cells or tissue samplesfor their content of the fibronectin receptor. Such analysis will beimportant in determining the adhesion capacity of cells such as those intumors. Alternatively, the isolated receptor can be used to prepareantibodies or such assays.

A reagent consisting essentially of the cell surface glycoproteindescribed in the foregoing examples may be used in therapy to carryreagents to selected tissues.

The cell surface glycoprotein is also useful in assaying for receptorantibodies and, together with such antibodies, will permit establishmentof graft tissue assays for the receptor such as a radioimmunoassay orenzyme linked assay.

What is claimed is:
 1. An antibody that specifically binds to a cellsurface glycoprotein, wherein the glycoprotein: a. is a human cellsurface glycoprotein; b. has an apparent molecular weight of about140,000 Daltons when measured by SDS-polyacrylamide gel electrophoresison a 7.5% gel under reducing conditions; c. has an apparent molecularweight of about 140,000 Daltons and 120,000 Daltons when measured bySDS-polyacrylamide gel electrophoresis on a 7.5% gel under non-reducingconditions; and d. specifically binds with the cell attachment site infibronectin, and is elutable from the cell attachment site offibrornectin by contacting with a peptide consisting essentially of thesequence GLY-ARG-GLY-ASP-SER-PRO.